Rabbit polyclonal anti 53bp1 antibody

Here, 5–7 × 10⁵ cell/mL were continuously exposed to 100 nM CPT (Sigma, St. Louis, MO, USA) for 1 hr. The cells were spun on the slide, then fixed with 4% paraformaldehyde in PBS, after which the cells were permeabilized by 0.1% Triton X-100 in PBS, and non-specific binding was blocked by the Odyssey^® blocking buffer. Rabbit monoclonal antibody against γH2AX, Ser139 (CST, Danvers, MA, USA), rabbit polyclonal IgG antibody against RAD51 (H-92) (SantaCruz, Dallas, TX, USA), mouse monoclonal antibody against RAD51 (SantaCruz), and rabbit polyclonal anti-53BP1 antibody (Novus Biologicals, Centennial, CO, USA) were used to detect γH2AX and 53BP1 focus formation, respectively. Alexa Fluor 647 donkey anti-rabbit IgG (Thermo Fisher Scientific) and Alexa Fluor 647 donkey anti-mouse IgG (Thermo Fisher Scientific) were used as the secondary antibody. The nuclei were counter-stained using Hoechst 33342 (Thermo Fisher Scientific). Images were acquired using the Operetta High-Content Analysis System (PerkinElmer, Waltham, MA, USA). At least 300 cells were counted by eye in images opened in ImageJ 1.52p [63 (link)]. Positive foci formation of γH2AX, 53BP1, and RAD54L cells were determined by more than four foci per cell.